Follicle-stimulating hormone (FSH), secreted by anterior pituitary gonadotrophs, is recognized as a crucial regulator of male and female gonadal function; indeed, reproductive biology textbooks strongly emphasize its role in normal physiology (Simoni et al., 1997). FSH exerts its effects via a specific receptor, follicle-stimulating hormone receptor (FSHR), which belongs to the highly conserved family of G-protein-coupled receptors (Chrusciel et al., 2019). Traditional views hold that in females, FSHR is expressed in granulosa cells and controls the maturation of Graafian follicles, granulosa cell proliferation, and estrogen production (Simoni et al., 1997), while in males, FSHR is expressed in testicular Sertoli cells and regulates their metabolic functions, which are essential for proper spermatogenesis and germ cell survival (Asatiani et al., 2002). The large FSHR gene is located on chromosome 2p21 and comprises 10 exons (Minegishi et al., 1991).

However, accumulating evidence demonstrates that FSHR is also expressed in extragonadal tissues, such as endothelium (Stilley and Segaloff, 2018), monocytes (Robinson et al., 2010), developing placenta (Stilley et al., 2014), endometrium (Sacchi et al., 2018), malignant tissues (Papadimitriou et al., 2016), bone, adipose, and neural cells (Bhartiya and Patel, 2021). Intriguingly, blocking the interaction of Fsh with Fshr mitigates some degenerative disorders in mice, such as low bone mass (Sun et al., 2006), obesity (Liu et al., 2017), and neurocognitive decline (Xiong et al., 2022). Fshr was reported to be expressed in osteoclasts in vitro, and global Fsh or Fshr knockout resulted in an increase in bone mass by inhibiting bone resorption (Sun et al., 2006). Blocking the Fsh-Fshr interaction with either Fsh polyclonal or monoclonal specific antibodies triggered thermogenesis in adipose tissues, significantly reduced body weights (Liu et al., 2017), and reduced Alzheimer’s symptoms in mice (Xiong et al., 2022). Fshr expression is also found in the vasculature of tissues (Stilley et al., 2014) and is particularly high in solid tumors (Ghinea, 2018). Nevertheless, the relevance of its extragonadal expression and functions have been debated (Chrusciel et al., 2019; Kumar, 2014). Therefore, precisely defining the localization of Fshr expression remains an imperative challenge in FSH-FSHR biology due to the concern of the ‘non-specificity’ of available antibodies used to localize FSHR expression and the ‘quick turnover’ and ‘rapid degradation’ of Fshr transcripts (Chrusciel et al., 2019; Bhartiya and Patel, 2021; Tedjawirja et al., 2023). Thus, a suitable genetic approach to resolve this issue is warranted (Kumar, 2018).

To address this challenge, we can utilize a GFP reporter driven by the endogenous promoter of Fshr, which enables the visualization of the expression of low-abundance transcripts in more accurate and context-specific ways (Chudakov et al., 2005; Cheng et al., 1996), when other approaches, e.g., antibodies and RT-PCR, are limited in their ability to detect Fshr expression. Therefore, in this study, we employed CRISPR/Cas9-mediated technology to create a novel Fshr-ZsGreen reporter mouse model for precisely clarifying Fshr expression. We believe that this powerful approach, as a reliable readout of Fshr expression at the single-cell level, should allow us to accurately capture Fshr expression in real time in a tissue-specific manner. We also used other techniques to ensure the accuracy and reliability of the results. Our results challenge the current understanding of Fshr expression and refine that Fshr has a wider expression profile than previously thought. The findings of Fshr in reproductive and nonreproductive tissues/organs will provide significant insights into new roles of Fsh and Fshr in physiology and pathology and have implications for the development of new therapies for reproductive and nonreproductive disorders, particularly metabolic diseases and degenerative diseases.

To precisely define Fshr expression in mice, we utilized several complementary strategies, including Fshr endogenous promoter driving ZsGreen knockin reporter mice, immunofluorescence (IF) staining with antibodies against tissue/cell type-specific markers and Fshr, droplet digital RT-PCR (ddRT-PCR) and single RNA-fluorescence in situ hybridization (RNA-smFISH), to comprehensively examine Fshr expression.

Generation of Fshr-ZsGreen knockin reporter mice

CRISPR/Cas9-mediated homologous recombination was used to generate embryonic stem cell clones, in which a P2A-ZsGreen cassette was precisely inserted before the stop codon of the Fshr gene followed by the 3′ UTR of the Fshr allele, as described in the Methods (Figure 1A). This P2A-ZsGreen expression cassette under the control of endogenous Fshr regulatory elements ultimately generates ZsGreen proteins without disrupting Fshr expression. The 19 amino acids P2A sequences function to cause ribosomal ‘skipping’ during translation, resulting in a missing peptide bond and effectively separates the two proteins, e.g., Fshr and ZsGreen (Zhu et al., 2023; Trichas et al., 2008; Luke and Ryan, 2018). The injected zygotes were transferred into oviducts of Kunming pseudopregnant females to generate F0 mice. F0 mice with the expected genotype were confirmed by tail genomic DNA PCR, DNA sequencing, and Southern blotting (Figure 1B and C) and then mated with C57BL/6J mice to establish F1 heterozygous mice with the germline-transmitted transgene. F1 heterozygous mice were further genotyped by tail genomic PCR, DNA sequencing and Southern blotting (Figure 1B and C). The results from these tail genomic PCR, DNA sequencing, and Southern blots in both F0 and F1 pups demonstrated that the targeted P2A-ZsGreen cassette was accurately inserted into the designed site between exon 10 and the stop codon of the Fshr gene before the 3’ UTR. We maintained heterogeneous Fshr-ZsGreen mice and used homogeneous mice for experiments. These mice were genotyped using primer sets specific to P2A-ZsGreen, as shown in Figure 1D. Fshr and GFP are transcribed in a bicistronic mRNA but subsequently translated into two independent proteins rather than as a fusion protein. This design ensures unaffected Gαi3 transcription and function and simultaneous GFP expression as a reporter protein. All animals were fertile and showed normal behavior and no obvious abnormal phenotypes.

Figure 1

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Figure 1—source data 1

Gel images for genotyping PCR of mouse tail DNA.

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Figure 1—source data 2

Original TIFF images for Southern blots.

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Reproductive organs

As the reproductive system is well known to express Fshr, we first tested Fshr-ZsGreen expression in the ovary and testis to ensure ZsGreen expression driven by the endogenous Fshr promoter. In the ovary, we observed Fshr-ZsGreen expression in the different stages of follicles from primordial follicles to primary follicles, secondary follicles, and the corpus luteum (Figure 2A-b and e). In the ovarian/Graafian follicles, expression was observed in the oocytes, granulosa cells/follicle cells and theca (interna and externa) (Figure 2A-b, c, e, and f). We also found Fshr-ZsGreen expression in the ciliated epithelial cells in the oviduct (Figure 2A-d and h). Furthermore, we employed an antibody against Stra8 (Miyauchi et al., 2017) to perform IF staining to identify reproductive cells and observed the colocalization of Fshr-ZsGreen with Stra8 staining as a marker for reproductive cells (Figure 2A).

Figure 2

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In the testis, we found Fshr-ZsGreen expression and its colocalization with Stra8 (Heinrich et al., 2020) staining in the cells of seminiferous tubules (STs), including primary spermatocytes, Sertoli cells, and spermatids, and particularly in interstitial Leydig cells between STs (Figure 2B). Figure 2B-a shows an image of the whole sectioned testis. A representative ST is displayed at two magnifications (Figure 2B-b and c), demonstrating strong expression of Fshr-ZsGreen in Leydig cells, as indicated by empty white arrows. In addition, we also applied an antibody against Set to identify testis cells, whose expression was reported in multiple cell types of the mouse testis at different developmental stages (Dai et al., 2014). In Figure 2C, a representative image of ST with Leydig cells is shown at a lower magnification (×400) and a higher magnification (×1000) (Figure 2C-a and b), showing colocalization of Fshr-ZsGreen and Set staining in testis cells. We found strong Set staining in Fshr-ZsGreen-positive spermatogonia, as indicated by empty white arrowheads (Figure 2C-b). Fshr-ZsGreen was also observed in the arterioles of the testis (Figure 2C-c).

Because of RNA in situ hybridization as the gold standard method for visualizing RNA expression and localization in cells, tissue sections, and whole organs (Young et al., 2020; Levsky and Singer, 2003), we further carried out single RNA molecule-fluorescence in situ hybridization to confirm Fshr expression in the Leydig cells, using antisense probe for identifying Fshr expression in the sections of Fshr-ZsGreen and B6 mice, whereas its sense probe was used as a negative control (Figure 2C and D). Additionally, we also examined Fshr expression by IF in Leydig cell line TM3 (Mather, 1980) and found that the majority of TM3 cells stained for lower Fshr, only a few cells stained relatively higher Fshr (Figure 2E-a and b). Interestingly, we also noticed Fshr expression located in the nuclei (Figure 2E-c to h).

We then performed ddRT-PCR to compare Fshr expression in TM3 cells, testis, and ovaries of Fshr-ZsG and B6 mice at 3 months of age (Figure 2G-a, representative of three experiments). The results showed that Fshr expression was similar in the testes and ovaries of both types of mice, indicating that the insertion of P2A-ZsGreen did not disrupt Fshr expression in Fshr-ZsGreen mice. However, Fshr expression was significantly higher in the testes compared to the ovaries, by almost 40-fold. Interestingly, TM3 cells exhibited much lower Fshr expression levels than the testes and ovaries. In fact, the levels were approximately 145-fold lower than in the testes and 3.6-fold lower than in the ovaries.

Last, we also searched for FSHR expression in single-cell RNA-seq (scRNA-seq) databases DISCO (https://immunesinglecell.org/genepage/FSHR) and BioGPS (http://biogps.org/#goto=genereport&id=2492), showing FSHR expression in human Leydig cells (Appendix 1—figure 3).

Overall, we observed Fshr-ZsGreen expression in the reproductive system, demonstrating that ZsGreen is a reliable readout for Fshr expression. In addition to its sole expression in granulosa cells and Sertoli cells, as reported previously, our findings clearly reveal that Fshr is also expressed in other cell types in the reproductive system, particularly in Leydig cells.

Skeletal tissues

Fsh has been thought to have a direct role in bone (Sun et al., 2006) , therefore we next examined the expression pattern of Fshr in femoral sections, as shown in Figure 3A, under confocal fluorescence microscopy. The representative areas are presented at two magnifications of ×400 and ×1000. In the epiphyseal growth plate, we observed lower expression of Fshr in chondrocytes, as indicated by two dotted lines, compared to its expression in cells located in the sponge area above or under the dotted lines (Figure 3A-a). At higher magnification, Fshr was expressed in the columns of chondrocytes from the resting zone to the transformation zone (Figure 3A-e).

Figure 3

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In contrast, we found that Fshr was brightly expressed in osteoblasts and osteoclasts in the metaphyseal trabeculae, which were recognized based on nuclear DAPI staining - osteoblasts were stained with a single DAPI nucleus, while osteoclasts contained more than two DAPI-stained nuclei (Figure 3A-c and d). Similarly, we further observed that these cells on the surfaces of trabeculae in bone marrow and cortical bone also clearly expressed Fshr-ZsGreen, as shown in Figure 3A-e and f (trabeculae) and Figure 3A-g and h (cortex). Importantly, we noted Fshr-ZsGreen expression in osteocytes (indicated by empty white arrowheads, Figure 3A-d, g, and h), the most abundant cell in the skeleton. These were embedded in trabecular and cortical bone, as well as in the periosteum (P) (Figure 3A-h).

To confirm the identification of osteoblasts/osteocytes and osteoclasts that expressed Fshr, we performed IF staining using an antibody against osteocalcin, a marker of osteoblasts (Karsenty, 2017). As shown in Figure 1B, we observed colocalization of Fshr expression with osteocalcin staining in chondrocytes in the transforming zone, as indicated by white arrows (Figure 3B-a), in cuboid and nucleated osteoblasts and bone lining cells on the trabecular and endosteal surfaces (Figure 3B-b and c), cortical bone, and osteocytes within the mineralized matrix (Figure 3B-b, c, and e).

To identify osteoclasts, we performed fluorescence immunostaining with an antibody against Trap, an osteoclast marker (Lindunger et al., 1990). Osteoclasts were recognized by positive staining for TRAP with more than two DAPI-stained nuclei. Multinucleated Fshr-ZsGreen-positive cells were stained positively for Trap located on the surface of the resorptive bays or areas adjacent to the trabecular bone (Figure 3B-e and f).

To examine whether skeletal stem/progenitor cells express Fshr, we performed IF staining for stem markers with antibodies against CD34 or CD133 (Chan et al., 2015; Kuroda et al., 2014; Handgretinger et al., 2003; Handgretinger and Kuçi, 2013; Pozzobon et al., 2009). Using these well-known stem markers, we identified Fshr-ZsGreen-positive cells as stem/progenitor cells located in the bone marrow, growth plate/articular cartilage, and periosteum, as shown in Figure 2C-a, b, c, e, and d, respectively. These cells also featured an increased nuclear-cytoplasmic ratio, except for these cells on the trabecular surface and in the periosteum.

We also examined Fshr expression in osteocytes from DMP1-CreERT²⁺:Fshr^fl/fl mice (osteocyte-specific Fshr cKO) where the control mice (DMP1-CreERT^2-:Fshr^fl/fl) as a positive control. We noticed a significant drop of Fshr expression in osteocytes in Fshr cKO mice when tamoxifen was intraperitoneally administrated to the mice (Figure 3D). We also found an increased thickness of cortex triggered by Fshr cKO (Figure 3D - the left panels, the thickness indicated by dotted lines). The finding further demonstrated Fshr present in osteocytes, the largest cell population in bones, and the specificity of the Fshr antibody.

Adipose tissues

Because our previous works and others have provided evidence on the role of Fsh in adipose tissues, we then examined Fshr-ZsGreen expression in adipose tissues. As expected, we found Fshr-ZsGreen expression in adipocytes of the frozen sectioned inguinal WAT, as shown in the left panel of Figure 4A at a low magnification (×40), which was further confirmed under a higher magnification (×400) in the three representative areas, demonstrating that Fshr-ZsGreen was expressed in the cellular membranes of individual adipocytes (Figure 4A-a, b, and c).

Figure 4

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To confirm adipocyte identification, we performed IF with an antibody against mouse Ucp1 (Liu et al., 2017) to recognize adipocytes, showing that the majority of Fshr-ZsGreen-positive cells were stained positively for Ucp1, and found two types of Fshr⁺ adipocyte populations: one with colocalization of the two markers only in the membrane (indicated by empty white arrowheads) and another with two markers in both the membrane and cytoplasm (indicated by white arrows) (Figure 4B-a and b). In addition, we observed that Fshr-ZsGreen was expressed in arterioles in adipose tissue, denoted by white empty arrowheads in Figure 4B-c and a dotted circle in Figure 4B-d.

We further examined Fshr-ZsGreen expression in BAT. Fshr-ZsGreen was observed across the whole section of examined BAT at a lower magnification (Figure 3A-a). At a higher magnification (×400), three representative areas are presented, as shown in Figure 3A-b, c, and d, in which Fshr-ZsGreen was expressed not only in the cellular membranes but also in areas of cytoplasm close to the membranes, as indicated by white arrows (Figure 4C-b, c, and d).

Furthermore, we also performed IF with three antibodies against Ucp1, Th, and Peri to identify brown cells and peripheral fibers in the Fshr-positive section. We found that several areas in the section were strongly stained for Ucp1 at low magnification (×40), as shown in Figure 4D-a. Under higher magnifications of ×400 and ×1000, we colocalized Fshr-ZsGreen with Ucp1 in the three representative areas, in which some locations had higher Fshr-ZsGreen expression and others had higher Ucp1 expression (Figure 4D-a, b, and d).

To examine whether peripheral neural fibers express Fshr, we used antibodies against Th and Peri that can recognize peripheral neural fibers. We found that neural fibers stained positively and surrounded Fshr-ZsGreen-positive arterioles in BAT (Figure 4E-a and b). In addition, Fshr-ZsGreen was expressed in the nodes of Ranvier of TH-stained small neural fibers (indicated by empty red arrows, Figure 4E-e). We further confirmed Fshr-ZsGreen expression in peripheral neural fibers by IF with an antibody against peripherin (Peri), which is a type III intermediate filament protein found predominantly in peripheral nerves, specifically in sensory and autonomic neurons (Figure 4F). We noted colocalization of Fshr-ZsGreen and Peri staining in large neural fibers (Figure 4F-b, d, and e). Interestingly, we observed that both markers for peripheral neural fibers were also expressed in the cytoplasm of BAT cells, in which Fshr-ZsGreen was strongly expressed (Figure 4E-b, c, and f; Figure 4F-e, c, and f).

To further ensure Fshr expression in adipose tissues, we also performed IF staining with Fshr antibody in white and BAT adipose sections of B6 mice. The results showed the similar expression pattern as seen in Fshr-ZsGreen mice (Figure 4G).

Taken together, the above-described findings on Fshr-ZsGreen expression in reproductive, skeletal, and adipose tissues convincingly demonstrate that Fshr-ZsGreen is a reliable readout of Fshr expression. Furthermore, we identified Fshr expression in Leydig cells and follicles at different developmental stages, cells of osteoblast lineage, and peripheral nerve fibers. With confidence that Fshr-ZsGreen is a reliable readout, we used this powerful tool to further examine Fshr expression in other tissues and organs.

Heart and aorta

To examine Fshr expression in the cardiovascular system, we used the heart and aorta as key organs/tissues to detect Fshr-ZsGreen expression. As expected, we observed strong Fshr-ZsGreen expression in the myocardium (Figure 5A-a) and large muscular arteries (a representative is shown in Figure 5A-b). Then, we further confirmed Fshr-ZsGreen expression by IFs with two antibodies against α-SMA and EMCN (Chai et al., 2023) that recognize alpha smooth muscle actin of smooth muscle and endomucin of endothelial cells at higher magnifications. With IF staining for α-SMA, we imaged several areas of both heart and blood vessels, as shown in the left image of the upper panel (×40). In the heart, we observed that Fshr-ZsGreen was highly expressed in cardiomyocytes in longitudinal and transverse orientations of the myocardium, which were positively stained for α-SMA (Figure 6B-a, b, and c). At a magnification of ×1000, it was also expressed in the endothelial layer of arterioles between muscle fibers (Figure 5B-i).

Figure 5

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Figure 6

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In addition to the cardiomyocytes, we also observed Fshr-ZsGreen in α-SMA-stained smooth muscles and endothelial cells of large blood vessels above the heart (Figure 7B-d). Interestingly, we found Fshr-ZsGreen in adipose tissue around the blood vessels, and the adipocytes morphologically appeared to be brown adipose cells, as the majority of these brown-like cells were full of ZsGreen-positive cytoplasm, instead of single large fat droplets with Fshr-ZsGreen expression in cellular membranes (Figure 5B-c, d, e, and j). The adipose tissue was stained positively for α-SMA, suggesting that the ZsGreen⁺ structures costained for α-SMA are blood vessels within the beige tissues, which are indicated by white empty arrowheads (Figure 5B-e and j).

Figure 7

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Furthermore, we observed bright Fshr-ZsGreen expression with slightly weak staining for α-SMA in the layer of endothelial cells of the large vein in Figure 5B-f and h. Under the endothelial cells, a cluster of smooth muscles showed colocalization of Fshr-ZsGreen with positive staining for α-SMA (Figure 5B-f and h). In contrast to the vein, we observed a thin layer of endothelial cells (tunica intima) and stronger Fshr-ZsGreen expression with positive staining for α-SMA in the large artery (tunica media); Fshr-ZsGreen was also present in the cells in the tunica adventitia (Figure 5B-k). In frozen sections of hearts, we observed strong Fshr-ZsGreen expression in neural fiber clusters that were in adipose tissue, as shown in Figure 5B-g and m.

Using an antibody against EMCN, a marker for endothelial cells, we further confirmed Fshr-ZsGreen expression in the layer of endothelium (tunica intima), which showed visible positive staining for EMCN when imaged at ×400 and ×1000 (Figure 5C-a, b, and d). In addition, Fshr-ZsGreen⁺ cardiomyocytes and smooth muscles under the endothelial layer were positive for EMCN staining (Figure 5C-a, b, and c).

In addition to large blood vessels on the heart, we also took a close look at the ascending aorta. We obtained two types of sections with longitudinal and transverse orientations for IFs with the two antibodies as above. In both sections, we found that Fshr-ZsGreen⁺ smooth muscle fibers were strongly stained for α-SMA in the first layer of SM close to the endothelium, where the second layer of smooth muscle was relatively weak for staining with α-SMA (Figures 5D-a and 6B-e). Fshr-ZsGreen was also present at the endothelium, which was costained positively for α-SMA (Figure 6A-a and B-e). Using an anti-EMCN antibody, we noticed that positive staining was in the upper part of Fshr-ZsGreen⁺ endothelial cells facing the lumen of the examined blood vessels (Figure 5A-c and B-f). In addition to Fshr-ZsGreen expression in the endothelium of the tunica intima, it was also observed in the areas of subendothelial connective tissue and tunica media (smooth muscle), but these areas were not stained for EMCN (Figure 5A-c and B-f).

Other key tissues and organs (liver, pancreas, thyroid, skin and skeletal muscle, spleen, bone marrow, and brain)

With the power of Fshr-ZsGreen, we characterized Fshr-ZsGreen expression in the liver. We observed Fshr-ZsGreen expression in the hepatocytes and arteries inside the hepatocytes, which were positively stained for Col1a1 or CD31 (Wang et al., 2021; Ismail et al., 2003), respectively (Figure 7A and B). Although it is weakly expressed inside large nerve fibers, Fshr-ZsGreen is strongly expressed in small neural fibers and shows a costaining pattern with KCBMA1 (Simms and Zamponi, 2014), a marker used for the detection of peripheral nerve fibers (Figure 7C, indicated by white arrowhead, whereas large nerve fibers are indicated by white arrows).

We then examined Fshr-ZsGreen expression in the pancreas and found that it was expressed not only in acinar cells but also in islets of Langerhans at low and high magnifications (Figure 8A-a and b). We then confirmed the identification of α- and β-cells by IFs with antibodies against NG3, insulin, or glucagon. The imaging results clearly demonstrated that Fshr-ZsGreen was expressed in both α- and β-cells as well as acinar cells at ×400 and ×1000 magnifications, respectively (Figure 8A–C).

Figure 8

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Regarding Fshr-ZsGreen expression in the thyroid, we found Fshr-ZsGreen in both follicular cells and parafollicular cells (C-cells) at low and high magnifications (×400 and ×1000) (Figure 9A-a, b, c, d, and e). We further used an anti-Tshr receptor antibody to confirm Fshr-ZsGreen-positive cells, which showed that follicular cells were stained positively for Tshr in the nuclei, as indicated by dotted circles and white arrowheads, and C-cells were indicated by white arrows (Figure 9-b to e).

Figure 9

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Next, to examine Fshr-ZsGreen expression in the skin, we used two types of skin sections: thick skin from the tail and thin skin from the abdomen (Figure 10A-a and b). Although a dermis layer was not included in the image taken for a thick sample, we observed that Fshr-ZsGreen was expressed in hair follicle (HF) and sweat gland cells and fibroblasts in the dermis and hypodermis (Figure 10A-a). Similarly, Fshr-ZsGreen was present in HFs and fibroblasts in the dermis and keratinocytes in the epidermis of the thin skin (Figure 10A-b). As we detected Fshr-ZsGreen expression in HFs, we wondered whether stem cells in HFs express Fshr-ZsGreen. To address this question, we carried out IF with an antibody against CD34, a stem cell marker. Not surprisingly, we found that CD34 staining was colocalized with cells with Fshr-ZsGreen in Bulge as quiescent stem cells and in the dermal papilla (DP) and epidermis as active stem/progenitor cells, as shown in Figure 10B-a and b. Therefore, these results indicate Fshr-ZsGreen expression in stem/progenitor cells in the skin (Figure 10B-a and d).

Figure 10

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We then detected Fshr expression in skeletal muscle (gastrocnemius), followed by IF staining using three antibodies against α-SMA, PAX7, and TH for the identification of satellite cells and peripheral nerve fibrils. We observed Fshr-ZsGreen across the muscle sections at a lower magnification of ×40 (Figure 11a). At higher magnifications, we found that Fshr-ZsGreen was present in the muscle fibers, in which one type had higher Fshr-ZsGreen expression (indicated by white arrowheads) and another type had lower Fshr-ZsGreen expression (Figure 11-b to h). Fshr-ZsGreen was also highly expressed in satellite cells stained positively for either αSMA (Figure 11-b to e) or PAX7 (Figure 11-f and g) in both longitudinal and transverse sections, as indicated by white arrows. In addition, we detected Fshr-ZsGreen expression in the peripheral neural fibers identified by positive staining for TH, which were around vascular structures in the muscle tissue, as indicated by red arrowheads (Figure 11-h and i).

Figure 11

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To detect Fshr expression in immune cells, we examined Fshr-ZsGreen expression in the spleen and bone marrow using antibodies against CD11b and CD3, as integrin αM (CD11b) is expressed in myeloid-lineage cells such as monocytes/macrophages, neutrophils, eosinophils, and basophils and in lymphoid cells such as NK cells and B-1 cells (Springer, 1994), while CD3 marks T-cells (Chatenoud and Bluestone, 2007). We imaged frozen sections of the spleen under magnifications of ×40, ×400, and ×1000 and observed that Fshr-ZsGreen was highly expressed in trabeculae and cells in both red and white pulps (RP and WP). The Fshr-ZsGreen-positive cells were further identified by IFs with antibodies against CD11B or CD3, indicating that Fshr is expressed in immune cells, such as monocytes/macrophages, neutrophils, eosinophils, basophils, NK cells, B-cells, and T-cells (Figure 12A and B). In addition, we further confirmed Fshr-ZsGreen expression in macrophages of bone marrow, which were identified with anti-CD4/80 antibody, as shown in two representative images of BM-one from an area located in the center of bone marrow (Figure 12C-a) and another close to cortical bone (Figure 12C-b) at ×1000 magnification (empty white arrows indicate the colocalization of Fshr-ZsGreen and CD11B, CD3, or CD4/80 in the RPs, while white arrows point to their colocalization in the WPs).

Figure 12

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Finally, we examined Fshr-ZsGreen expression and its colocalization with markers for either astrocytes, microglia, or neurons in the brain. As expected, we observed Fshr expression across the brain sections and the three representatives from the olfactory bulbs, pallidum, and hippocampus (CA3) are shown in Figure 12, demonstrating that Fshr is indeed expressed in astrocytes (Figure 13-a to c), microglia (Figure 12-d to f), and neurons (Figure 13-g to j), as well as neuronal fibers (synapses or projections). Other cell types are needed to be further defined.

Figure 13

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Confirmation of Fshr-ZsGreen expression with an Fshr-specific antibody and ddRT-PCR

Finally, to confirm the accuracy of the above results obtained from Fshr-ZsGreen mice, we performed fluorescence immunostaining with a specific antibody against mouse Fshr and accurate ddRT-PCR with mouse Fshr-specific primers to confirm the above data. An isotype-matched rabbit IgG was used as a negative control for IFs with anti-Fshr antibody using sections from Fshr-ZsGreen mice. Imaging was performed under the same conditions to record each corresponding tissue/organ stained with anti-Fshr antibody. The images of negative controls are shown in Appendix 1—figure 2, showing specific binding of the secondary antibody to anti-Fshr antibody without any nonspecific binding of the secondary antibody to the examined sections.

As shown in Figure 14A, in all the examined tissues, including bone, BAT, thyroid, cardiac muscle, kidney, liver, lung, aorta, ovary, and testis, we observed colocalization of Fshr-ZsGreen with positive staining for Fshr, further confirming the specific expression of Fshr-ZsGreen in the examined tissues/organs.

Figure 14

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We also obtained total RNA from the following tissues: lung, kidney, thoracic vertebra, calvaria, femur, jejunum, liver, teeth, tibia, skeletal muscle, tails, cartilage, skin, spleen, stomach, heart, bladder, tongue, BAT, WAT, thyroid, brain, pancreas, and duodenum. After reversing mRNA from total RNA, we performed ddRT-PCR with mouse-specific primers to check Fshr expression at the transcriptional level as described in the Methods section. The results demonstrated Fshr expression in these tissues, and the expression profile was categorized into three groups: (1) high expression in the lung and kidney tissues; (2) moderate expression in the thoracic (T) vertebrae, calvaria, femur, jejunum, liver, teeth, tibia, and muscle; and (3) low expression in the tail, cartilage, skin, spleen, stomach, heart, bladder, tongue, BAT, WAT, brain, pancreas, and duodenum (Figure 14B).

Finally, we also searched Fshr expression at single-cell level in four single-cell databases including DISCO (a database of Deeply Integrated Single-Cell Omics data, https://www.immunesinglecell.org/), BioGPS (a free extensible and customizable gene annotation portal, a complete resource for gene expression and protein function, http://biogps.org/exrna/#goto=welcome), Single Cell Portal (SCP, an interactive home for single-cell genomics data, https://singlecell.broadinstitute.org/), and Genotype-Tissue Expression (GTEx portal, https://gtexportal.org). The selected results from these databases are presented in Appendix 1—figure 3, which further support our findings of the widespread express pattern of Fshr described as above. In particular, Fshr expression was detected in Leydig cells by scRNA-seq as seen in DISCO (https://immunesinglecell.org/genepage/FSHR) and BioGPS (http://biogps.org/#goto=genereport&id=2492) for human cells.

Taken together, these data further confirmed Fshr-ZsGreen expression patterns from the Fshr-ZsGreen reporter line, convincingly demonstrating that Fshr is not limited to previously reported cells, tissues, or organs, such as the reproductive system, osteoclasts, adipose, endothelium in tumors, and neurons in the brain, but rather has a wide expression in the cells, tissues, and organs in the body, particularly in the lung, kidney, and heart, as well as Leydig cells in the testis, which has not previously been recognized.

Although numerous efforts have been made to characterize Fshr expression in tissues/cells, defining the locations of Fshr expression remains an imperative challenge in Fsh-Fshr biology, primarily because of concerns about the specificity of available antibodies against Fshr (Chrusciel et al., 2019; Kumar, 2014; Tedjawirja et al., 2023). In this case, we developed CRISPR/Cas9-mediated Fshr-ZsGreen knockin reporter mice to address this issue.

To maintain the integrity of the splicing acceptor, donor, and gene promoter regions, we designed and inserted the ZsGreen (ZsG) reporter into the C-terminus of Fshr by sequence-specific gRNA-guided CRISPR/Cas9-mediated precise genome modification with a long ssDNA template to create the Fshr-P2A-ZsG reporter mice (Bai et al., 2020). Because of its high turnover of Fshr mRNA and a difficulty in detection of Fshr expression by RT-PCR and northern blotting, in this reporter line, we utilized ZsG as a GFP reporter, as ZsGreen, also called ZsGreen1, is an exceptionally bright green fluorescent protein derived from a Zoanthus sp. reef coral (Matz et al., 1999) that has been modified for high solubility, bright emission, and rapid chromophore maturation. ZsGreen is the brightest commercially available green fluorescent protein - up to 4× brighter than EGFP with the half-life of 26 hr - and is ideally suited for whole-cell labeling and promoter-reporter studies to indicate the promoter activity of a gene of interest, which has been used in numerous GFP reporter mice (Zhu et al., 2012b; Pippin et al., 2013; Pippin et al., 2014; Büller et al., 2015; Dewas et al., 2015; Nazareth et al., 2015; Xiao et al., 2015; Kaverina et al., 2016; Saito et al., 2016; Fougère et al., 2021). In addition, we also employed a short and conserved picornavirus-derived ‘self-cleaving’ 2A peptide to allow bicistronic expression of Fshr and ZsG, as the 19-amino acid P2A has the highest cleavage efficiency (Wang et al., 2015; Kim et al., 2011). The cleavage is triggered by ribosomal skipping of the peptide bond between the proline (P) and glycine (G) in C-terminal of P2A peptide, resulting in the peptide located upstream of the 2A peptide to have extra amino acids on its C-terminal end, while the peptide located downstream the 2A peptide will have an extra proline on its N-terminal end. Therefore, P2A can nearly equalize the expression of genes upstream and downstream. As a result, Fshr is normally expressed without interruptions and ZsG expression indicates Fshr promoter activity in the defined cells, demonstrated by the comparison of Fshr expression in the testes and ovaries between Fshr-ZsGreen and B6 mice (Figure 2G).

The P2A-ZsGreen construct was precisely inserted into the site between the last exon (exon 10) and the stop codon of the Fshr locus, which was confirmed by integration detection PCR, sequencing of PCR fragments, and Southern blotting with Fshr locus-specific enzyme restriction digestions. Successful insertion allows the endogenous promoter of Fshr to drive ZsGreen reporter expression. Because of the site-specific insertion of the P2A ZsG vector to produce Fshr-ZsG reporter, we only used one founder line for characterizing Fshr-ZsG expression, rather than multiple founders when random insertions of a transgene were used previously for generating transgenic mice.

This approach greatly enhanced our understanding of this critical cellular pathway in several ways. First, employing the native regulatory element responsible for governing the expression of the target gene ensures that the expression pattern of the GFP reporter closely mirrors that of the gene of interest, Fshr, within its natural context. Consequently, this approach provides a more faithful representation of gene expression. Second, endogenous promoters often exhibit specific spatial and temporal expression patterns, driving gene expression in specific cell types or developmental stages. This capability facilitates the capture of the dynamic nature of gene regulation and enables the study of gene expression under diverse physiological conditions. Third, utilizing endogenous promoters minimizes potential perturbations to the native gene regulation machinery. It avoids the need to introduce exogenous elements or artificial constructs, reducing the likelihood of altering the gene’s expression behavior or interfering with its regulatory interactions. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the target’s activity in live cells. Therefore, these distinct advantages of enhanced physiological relevance, precise spatiotemporal control, preservation of regulatory elements, and minimal perturbation provide us with a powerful tool to understand Fshr expression in more accurate and context-specific ways compared to other methods, such as using antibodies, northern blotting, RT-PCR, and in situ hybridization.

In this study, we systemically investigated Fshr expression at the single-cell level in this reporter line with confocal fluorescence microscopy and further confirmed the location of Fshr-ZsGreen expression by IF staining, in situ hybridization, and ddRT-PCR. The results from this work demonstrate that as a receptor for Fsh, Fshr is widely expressed in virtually every cell at variable levels in the examined tissues/organs of mice.

As expected in the examined testis, we noticed Fshr expression in Sertoli cells in the testis and granular cells in the ovary. Surprisingly, we observed that Fshr was also more strongly expressed in Leydig cells than in Sertoli cells. This expression was also detected in spermatocytes, spermatids, spermatozoa, and spermatogonia. Although this finding is different from present thoughts that Fshr is only present in Sertoli cells but not in other cell types of the testis, our finding is in line with previous works in fishes (including African catfish, zebrafish, teleosts, and Japanese eels) (García-López et al., 2009; Levavi-Sivan et al., 2010; García-López et al., 2010; Burow et al., 2020; Chauvigné et al., 2012; Ohta et al., 2007, rats An et al., 2022), dogs (Kasimanickam and Kasimanickam, 2015), and humans (Bonci et al., 2018) performed by immunohistochemistry (IHC) and in situ hybridization. In humans, Fshr was also highly expressed in Leydig cells, although it was taken as non-specificity of the anti-Fshr antibodies. However, as shown in this study, this is not the case, because Fshr is widely expressed, as demonstrated in our work.

Furthermore, our findings are supported by a report of the failure of normal Leydig cell development resulting from the deficiency of Fshr but not Fsh-beta (Baker et al., 2003) and scRNA-seq studies in Hu sheep (originated from Mongolian sheep) (Su et al., 2023) and in human as shown in Appendix 1—figure 3 (DISCO, https://immunesinglecell.org/genepage/FSH) and BioGPS for human cells (http://biogps.org/#goto=genereport&id=2492). Fshr expression in Leydig cells strongly indicates that Fsh-Fshr plays a role in the production of steroids in males. Unexpectedly, Leydig cell line TM3 expresses much lower Fshr, compared to the Leydig cells in vivo (Figure 2B–G). It may provide an evidence on a reason that these cells do not respond well to FSH treatments (Mather, 1980), and indicates that this cell line may not be a typical Leydig cell population and new Leydig cell lines should be established in the future.

In the ovary, Fshr is highly expressed in follicles at different stages, from primordial cells, primary follicles, and secondary follicles to mature/Graafian follicles and the corpus luteum. In addition to granulosa cells, Fshr expression was observed in oocytes of follicles. Taken together, these data indicate that Fshr plays an intragonadal role in the ovary and testis beyond the granulosa and Sertoli cells.

In the skeletal system, we observed Fshr expression in vivo not only in osteoclasts, as reported previously (Sun et al., 2006; Zhu et al., 2012a; Zhu et al., 2012c), but also, interestingly, in osteoblast lineage cells, such as osteoblasts, bone lining cells, osteocytes, and progenitor cells of the periosteum, as well as in chondrocytes. In previous reports, Fshr expression in osteoclasts was detected only by RT-PCR, western blot, and immunostaining in cultures of primary murine precursors (Sun et al., 2006; Zhu et al., 2012a; Zhu et al., 2012c). Using the Fshr-ZsGreen reporter line, we visualized Fshr expression in multinucleated osteoclasts in frozen sections, clearly demonstrating Fshr expression in osteoclasts. Intriguingly, this powerful tool enabled us to examine Fshr expression in other cell types in bone. Surprisingly, we observed Fshr expression in cells of the osteoblast lineage, from osteoprogenitor cells and osteoblasts to osteocytes and bone lining cells. This finding indicates that Fsh may regulate not only osteoclast-mediated bone resorption but also osteoblasts for bone formation. To functionally prove the presence of Fshr in osteoblasts/osteocytes, we also deleted Fshr in osteocytes in an inducible model. The Fshr cKO induced in osteocytes significantly reduce Fshr expression and triggered an increase in the cortical thickness (Figure 3D) and a much more profound increase in bone mass and a decrease in fat mass than blockade by Fsh antibodies (unpublished data), illuminating Fshr expression in the osteoblast lineage.

In addition to its expression in the reproductive system and skeletal system, we also strikingly identified other cell types that highly express Fshr: endothelial cells in blood vessels and epithelial cells in the lung and kidney. In every examined tissue/organ, we found that endothelial cells stained positively for CD34 lining on the arterioles had a higher expression of Fshr-ZsGreen than other cell types. This bright Fshr-ZsGreen is more obviously seen in large arteries, such as the ascending aorta and others located in the heart. Similarly, Fshr was detected in vessels in solid malignant tumors by IHC or RT-PCR (Abudureyimu et al., 2018). However, it was not seen in normal tissues or organs by these methods, possibly due to its rapid turnover, fast degradation, or selected antibodies. Strikingly, we detected the highest Fshr-ZsGreen expression in bronchial and bronchiole ciliated epithelial cells by both fluorescence microscopy and ddRT-PCR. It was also more highly expressed in other cell types in the lung, such as type I pneumocytes (alveolar lining cells), type II pneumocytes (great alveolar or septal cells), and gland cells. However, the role of this unexpectedly high Fshr expression in the lung remains unknown. Similarly, we also found the second highest expression of Fshr-ZsGreen in renal epithelial cells in proximal and distal convoluted tubules but weak expression in renal corpuscles. In addition, our finding of Fshr expression in adipose tissues is consistent with the observations from previous works (Liu et al., 2017; Liu et al., 2015). Recently, Fshr expression was reported to be present in β-cells of the pancreas to regulate glucose-stimulated insulin secretion, further supporting our findings of the Fshr expression pattern (Cheng et al., 2024).

In summary, we established and validated an Fshr-ZsGreen protein reporter in vivo that faithfully recapitulates endogenous Fshr expression at single-cell resolution. Our compelling findings reveal that in addition to gonadal tissues, Fshr is also highly expressed in extragonadal systems, such as the lung, kidney, heart, and pancreas. This will provide insight to better understand the biology of the Fsh-Fshr axis and its roles in the physiology and pathology of these tissues/organs. In addition to the above described, we detected Fshr expression in cells of the teeth and brain; those findings are not presented here because of space limitations and will be published elsewhere, except that three representative areas of the brain are shown in Figure 14.

Although the Fshr-ZsGreen reporter line is a powerful tool for detecting the location of Fshr expression, it is limited in the definition of individual isoforms of Fshr transcripts and the detection of their turnover, which should be addressed by specific antibodies and determined by the rates of transcription and RNA degradation, including Xrn1 for 5'-to-3' degradation, exosomes for 3'-to-5' degradation, and nonsense-mediated decay (Wada and Becskei, 2017; Kaverina et al., 2016).

The sequences of sense and antisense probes for FISH in testis:

1. For the sense probe:

   • 5'-ATCACTGGCTGTGTCATTGCTCTAA-3'

2. For antisense probes: a mixture of the following 5 probes used for FISH.

   • 5'-TTAGAGCAATGACACAGCCAGTGAT-3'

   • 5'-CACCTTGCTATCTTGGCAGAGGAAG-3

   • '5'-AGCACAAATCTCAGTTCAATGGCGT-3'

   • 5'-CAAGTGTTTAATGCCTGTGTTGGAT-3'

   • 5'-GCAGGGAATAGACCTTTGTCCTTGA-3'

Raw dataset presented in this study were deposited into a repository Dryad, including gel images of genotyping PCR and Southern blots; confocal images of Fshr-ZsGreen expression and immunofluorescence staining with various antibodies for tissue/cell type markers of frozen sections of organs harvested from Fshr-ZsGreen reporter and B6 mice, and smFISH; and dd.rtPCR for Fshr expression of mouse organs and TM3 cells.

1. 1. Liu P

   (2024) Dryad Digital Repository

   Atlas of Fshr Expression from Novel Reporter Mice.

   https://doi.org/10.5061/dryad.1jwstqk4t

1. 1. Abudureyimu A
   2. Cai Y
   3. Huo S
   4. Ren R
   5. Zang R
   6. Yang J
   7. Ma Z
   8. Cui Y

   (2018) Expression and localization of follicle-stimulating hormone receptor in the yak uterus during different stages of the oestrous cycle

   Reproduction in Domestic Animals = Zuchthygiene 53:1539–1545.

   https://doi.org/10.1111/rda.13313

   • PubMed
   • Google Scholar
2. 1. An K
   2. Yao B
   3. Kang Y
   4. Bao M
   5. Tan Y
   6. Pu Q
   7. Su J

   (2022) Seasonal expression of gonadotropin genes in the pituitary and testes of male plateau zokor (Eospalax baileyi)

   Animals 12:725.

   https://doi.org/10.3390/ani12060725

   • PubMed
   • Google Scholar
3. 1. Asatiani K
   2. Gromoll J
   3. Eckardstein SV
   4. Zitzmann M
   5. Nieschlag E
   6. Simoni M

   (2002) Distribution and function of FSH receptor genetic variants in normal men

   Andrologia 34:172–176.

   https://doi.org/10.1046/j.1439-0272.2002.00493.x

   • PubMed
   • Google Scholar
4. 1. Bai H
   2. Liu L
   3. An K
   4. Lu X
   5. Harrison M
   6. Zhao Y
   7. Yan R
   8. Lu Z
   9. Li S
   10. Lin S
   11. Liang F
   12. Qin W

   (2020) CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish

   BMC Genomics 21:67.

   https://doi.org/10.1186/s12864-020-6493-4

   • PubMed
   • Google Scholar
5. 1. Baker PJ
   2. Pakarinen P
   3. Huhtaniemi IT
   4. Abel MH
   5. Charlton HM
   6. Kumar TR
   7. O’Shaughnessy PJ

   (2003) Failure of normal Leydig cell development in follicle-stimulating hormone (FSH) receptor-deficient mice, but not FSHbeta-deficient mice: role for constitutive FSH receptor activity

   Endocrinology 144:138–145.

   https://doi.org/10.1210/en.2002-220637

   • PubMed
   • Google Scholar
6. 1. Bhartiya D
   2. Patel H

   (2021) An overview of FSH-FSHR biology and explaining the existing conundrums

   Journal of Ovarian Research 14:144.

   https://doi.org/10.1186/s13048-021-00880-3

   • Google Scholar
7. 1. Bonci EA
   2. Buiga R
   3. Badan M
   4. Iuliana Maja L
   5. Gata VA
   6. Lisencu IC
   7. Irimie A
   8. Achimas Cadariu P
   9. Piciu D

   (2018)

   Follicle-stimulating hormone receptors: a comparison of commercially-available monoclonal and polyclonal antibodies as immunohistochemical markers for cancer research

   Journal of BUON 23:1912–1921.

   • PubMed
   • Google Scholar
8. 1. Büller NVJA
   2. Rosekrans SL
   3. Metcalfe C
   4. Heijmans J
   5. van Dop WA
   6. Fessler E
   7. Jansen M
   8. Ahn C
   9. Vermeulen JLM
   10. Westendorp BF
   11. Robanus-Maandag EC
   12. Offerhaus GJ
   13. Medema JP
   14. D’Haens GRAM
   15. Wildenberg ME
   16. de Sauvage FJ
   17. Muncan V
   18. van den Brink GR

   (2015) Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice

   Gastroenterology 148:170–180.

   https://doi.org/10.1053/j.gastro.2014.10.006

   • PubMed
   • Google Scholar
9. 1. Burow S
   2. Mizrahi N
   3. Maugars G
   4. von Krogh K
   5. Nourizadeh-Lillabadi R
   6. Hollander-Cohen L
   7. Shpilman M
   8. Atre I
   9. Weltzien F-A
   10. Levavi-Sivan B

   (2020) Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes

   General and Comparative Endocrinology 285:113276.

   https://doi.org/10.1016/j.ygcen.2019.113276

   • PubMed
   • Google Scholar
10. 1. Chai W
    2. Hao W
    3. Liu J
    4. Han Z
    5. Chang S
    6. Cheng L
    7. Sun M
    8. Yan G
    9. Liu Z
    10. Liu Y
    11. Zhang G
    12. Xing L
    13. Chen H
    14. Liu P

    (2023) Visualizing Cathepsin K-cre expression at the single-cell level with GFP Reporters

    JBMR Plus 7:e10706.

    https://doi.org/10.1002/jbm4.10706

    • PubMed
    • Google Scholar
11. 1. Chan CKF
    2. Seo EY
    3. Chen JY
    4. Lo D
    5. McArdle A
    6. Sinha R
    7. Tevlin R
    8. Seita J
    9. Vincent-Tompkins J
    10. Wearda T
    11. Lu W-J
    12. Senarath-Yapa K
    13. Chung MT
    14. Marecic O
    15. Tran M
    16. Yan KS
    17. Upton R
    18. Walmsley GG
    19. Lee AS
    20. Sahoo D
    21. Kuo CJ
    22. Weissman IL
    23. Longaker MT

    (2015) Identification and specification of the mouse skeletal stem cell

    Cell 160:285–298.

    https://doi.org/10.1016/j.cell.2014.12.002

    • PubMed
    • Google Scholar
12. 1. Chatenoud L
    2. Bluestone JA

    (2007) CD3-specific antibodies: a portal to the treatment of autoimmunity

    Nature Reviews. Immunology 7:622–632.

    https://doi.org/10.1038/nri2134

    • PubMed
    • Google Scholar
13. 1. Chauvigné F
    2. Verdura S
    3. Mazón MJ
    4. Duncan N
    5. Zanuy S
    6. Gómez A
    7. Cerdà J

    (2012) Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost

    Biology of Reproduction 87:35.

    https://doi.org/10.1095/biolreprod.112.100784

    • PubMed
    • Google Scholar
14. 1. Cheng L
    2. Fu J
    3. Tsukamoto A
    4. Hawley RG

    (1996) Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells

    Nature Biotechnology 14:606–609.

    https://doi.org/10.1038/nbt0596-606

    • PubMed
    • Google Scholar
15. 1. Cheng Y
    2. Zhu H
    3. Ren J
    4. Wu H-Y
    5. Yu J-E
    6. Jin L-Y
    7. Pang H-Y
    8. Pan H-T
    9. Luo S-S
    10. Yan J
    11. Dong K-X
    12. Ye L-Y
    13. Zhou C-L
    14. Pan J-X
    15. Meng Z-X
    16. Yu T
    17. Jin L
    18. Lin X-H
    19. Wu Y-T
    20. Yang H-B
    21. Liu X-M
    22. Sheng J-Z
    23. Ding G-L
    24. Huang H-F

    (2024) Follicle-stimulating hormone orchestrates glucose-stimulated insulin secretion of pancreatic islets

    Nature Communications 14:6991.

    https://doi.org/10.1038/s41467-023-42801-6

    • Google Scholar
16. 1. Chrusciel M
    2. Ponikwicka-Tyszko D
    3. Wolczynski S
    4. Huhtaniemi I
    5. Rahman NA

    (2019) Extragonadal FSHR expression and function-is it real?

    Frontiers in Endocrinology 10:32.

    https://doi.org/10.3389/fendo.2019.00032

    • PubMed
    • Google Scholar
17. 1. Chudakov DM
    2. Lukyanov S
    3. Lukyanov KA

    (2005) Fluorescent proteins as a toolkit for in vivo imaging

    Trends in Biotechnology 23:605–613.

    https://doi.org/10.1016/j.tibtech.2005.10.005

    • PubMed
    • Google Scholar
18. 1. Dai X-N
    2. Liu S
    3. Shao L
    4. Gao C
    5. Gao L
    6. Liu J-Y
    7. Cui Y-G

    (2014) Expression of the SET protein in testes of mice at different developmental stages

    Asian Journal of Andrology 16:689–693.

    https://doi.org/10.4103/1008-682X.129937

    • PubMed
    • Google Scholar
19. 1. Dewas C
    2. Chen X
    3. Honda T
    4. Junttila I
    5. Linton J
    6. Udey MC
    7. Porcella SF
    8. Sturdevant DE
    9. Feigenbaum L
    10. Koo L
    11. Williams J
    12. Paul WE

    (2015) TSLP expression: analysis with a ZsGreen TSLP reporter mouse

    Journal of Immunology 194:1372–1380.

    https://doi.org/10.4049/jimmunol.1400519

    • PubMed
    • Google Scholar
20. 1. Fougère M
    2. van der Zouwen CI
    3. Boutin J
    4. Ryczko D

    (2021) Heterogeneous expression of dopaminergic markers and Vglut2 in mouse mesodiencephalic dopaminergic nuclei A8-A13

    The Journal of Comparative Neurology 529:1273–1292.

    https://doi.org/10.1002/cne.25020

    • PubMed
    • Google Scholar
21. 1. García-López A
    2. Bogerd J
    3. Granneman JCM
    4. van Dijk W
    5. Trant JM
    6. Taranger GL
    7. Schulz RW

    (2009) Leydig cells express follicle-stimulating hormone receptors in African catfish

    Endocrinology 150:357–365.

    https://doi.org/10.1210/en.2008-0447

    • PubMed
    • Google Scholar
22. 1. García-López A
    2. de Jonge H
    3. Nóbrega RH
    4. de Waal PP
    5. van Dijk W
    6. Hemrika W
    7. Taranger GL
    8. Bogerd J
    9. Schulz RW

    (2010) Studies in zebrafish reveal unusual cellular expression patterns of gonadotropin receptor messenger ribonucleic acids in the testis and unexpected functional differentiation of the gonadotropins

    Endocrinology 151:2349–2360.

    https://doi.org/10.1210/en.2009-1227

    • PubMed
    • Google Scholar
23. 1. Ghinea N

    (2018) Vascular endothelial FSH receptor, a target of interest for cancer therapy

    Endocrinology 159:3268–3274.

    https://doi.org/10.1210/en.2018-00466

    • PubMed
    • Google Scholar
24. 1. Guo X
    2. Sunil C
    3. Adeyanju O
    4. Parker A
    5. Huang S
    6. Ikebe M
    7. Tucker TA
    8. Idell S
    9. Qian G

    (2024) PD-L1 mediates lung fibroblast to myofibroblast transition through Smad3 and β-catenin signaling pathways

    Scientific Reports 12:3053.

    https://doi.org/10.1038/s41598-022-07044-3

    • Google Scholar
25. 1. Handgretinger R
    2. Gordon PR
    3. Leimig T
    4. Chen X
    5. Buhring H-J
    6. Niethammer D
    7. Kuci S

    (2003) Biology and plasticity of CD133+ hematopoietic stem cells

    Annals of the New York Academy of Sciences 996:141–151.

    https://doi.org/10.1111/j.1749-6632.2003.tb03242.x

    • PubMed
    • Google Scholar
26. 1. Handgretinger R
    2. Kuçi S

    (2013) CD133-positive hematopoietic stem cells: from biology to medicine

    Advances in Experimental Medicine and Biology 777:99–111.

    https://doi.org/10.1007/978-1-4614-5894-4_7

    • PubMed
    • Google Scholar
27. 1. Heinrich A
    2. Potter SJ
    3. Guo L
    4. Ratner N
    5. DeFalco T

    (2020) Distinct roles for Rac1 in sertoli cell function during testicular development and spermatogenesis

    Cell Reports 31:107513.

    https://doi.org/10.1016/j.celrep.2020.03.077

    • PubMed
    • Google Scholar
28. 1. Huhtaniemi IT
    2. Eskola V
    3. Pakarinen P
    4. Matikainen T
    5. Sprengel R

    (1992) The murine luteinizing hormone and follicle-stimulating hormone receptor genes: transcription initiation sites, putative promoter sequences and promoter activity

    Molecular and Cellular Endocrinology 88:55–66.

    https://doi.org/10.1016/0303-7207(92)90009-u

    • PubMed
    • Google Scholar
29. 1. Ismail JA
    2. Poppa V
    3. Kemper LE
    4. Scatena M
    5. Giachelli CM
    6. Coffin JD
    7. Murry CE

    (2003) Immunohistologic labeling of murine endothelium

    Cardiovascular Pathology 12:82–90.

    https://doi.org/10.1016/s1054-8807(02)00166-7

    • PubMed
    • Google Scholar
30. 1. Karsenty G

    (2017) update on the biology of osteocalcin

    Endocrine Practice 23:1270–1274.

    https://doi.org/10.4158/EP171966.RA

    • PubMed
    • Google Scholar
31. 1. Kasimanickam VR
    2. Kasimanickam RK

    (2015) Sertoli, leydig, and spermatogonial cells’ specific gene and protein expressions as dog testes evolve from immature into mature states

    Animals 12:271.

    https://doi.org/10.3390/ani12030271

    • Google Scholar
32. 1. Kaverina NV
    2. Eng DG
    3. Schneider RRS
    4. Pippin JW
    5. Shankland SJ

    (2016) Partial podocyte replenishment in experimental FSGS derives from nonpodocyte sources

    American Journal of Physiology. Renal Physiology 310:F1397–F1413.

    https://doi.org/10.1152/ajprenal.00369.2015

    • PubMed
    • Google Scholar
33. 1. Kim JH
    2. Lee S-R
    3. Li L-H
    4. Park H-J
    5. Park J-H
    6. Lee KY
    7. Kim M-K
    8. Shin BA
    9. Choi S-Y

    (2011) High cleavage efficiency of a 2A peptide derived from porcine Teschovirus-1 in human cell lines, Zebrafish and Mice

    PLOS ONE 6:e18556.

    https://doi.org/10.1371/journal.pone.0018556

    • Google Scholar
34. 1. Kumar TR

    (2014) Extragonadal FSH receptor: is it real?1

    Biology of Reproduction 91:99.

    https://doi.org/10.1095/biolreprod.114.124222

    • Google Scholar
35. 1. Kumar TR

    (2018) Extragonadal actions of FSH: a critical need for novel genetic models

    Endocrinology 159:2–8.

    https://doi.org/10.1210/en.2017-03118

    • Google Scholar
36. 1. Kuroda R
    2. Matsumoto T
    3. Kawakami Y
    4. Fukui T
    5. Mifune Y
    6. Kurosaka M

    (2014) Clinical impact of circulating CD34-positive cells on bone regeneration and healing

    Tissue Engineering. Part B, Reviews 20:190–199.

    https://doi.org/10.1089/ten.TEB.2013.0511

    • PubMed
    • Google Scholar
37. 1. Levavi-Sivan B
    2. Bogerd J
    3. Mañanós EL
    4. Gómez A
    5. Lareyre JJ

    (2010) Perspectives on fish gonadotropins and their receptors

    General and Comparative Endocrinology 165:412–437.

    https://doi.org/10.1016/j.ygcen.2009.07.019

    • Google Scholar
38. 1. Levsky JM
    2. Singer RH

    (2003) Fluorescence in situ hybridization: past, present and future

    Journal of Cell Science 116:2833–2838.

    https://doi.org/10.1242/jcs.00633

    • PubMed
    • Google Scholar
39. 1. Lindunger A
    2. MacKay CA
    3. Ek-Rylander B
    4. Andersson G
    5. Marks SC Jr

    (1990) Histochemistry and biochemistry of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid adenosine triphosphatase (TrATPase) in bone, bone marrow and spleen: implications for osteoclast ontogeny

    Bone and Mineral 10:109–119.

    https://doi.org/10.1016/0169-6009(90)90086-u

    • PubMed
    • Google Scholar
40. 1. Liu X-M
    2. Chan HC
    3. Ding G-L
    4. Cai J
    5. Song Y
    6. Wang T-T
    7. Zhang D
    8. Chen H
    9. Yu MK
    10. Wu Y-T
    11. Qu F
    12. Liu Y
    13. Lu Y-C
    14. Adashi EY
    15. Sheng J-Z
    16. Huang H-F

    (2015) FSH regulates fat accumulation and redistribution in aging through the Gαi/Ca(2+)/CREB pathway

    Aging Cell 14:409–420.

    https://doi.org/10.1111/acel.12331

    • PubMed
    • Google Scholar
41. 1. Liu P
    2. Ji Y
    3. Yuen T
    4. Rendina-Ruedy E
    5. DeMambro VE
    6. Dhawan S
    7. Abu-Amer W
    8. Izadmehr S
    9. Zhou B
    10. Shin AC
    11. Latif R
    12. Thangeswaran P
    13. Gupta A
    14. Li J
    15. Shnayder V
    16. Robinson ST
    17. Yu YE
    18. Zhang X
    19. Yang F
    20. Lu P
    21. Zhou Y
    22. Zhu L-L
    23. Oberlin DJ
    24. Davies TF
    25. Reagan MR
    26. Brown A
    27. Kumar TR
    28. Epstein S
    29. Iqbal J
    30. Avadhani NG
    31. New MI
    32. Molina H
    33. van Klinken JB
    34. Guo EX
    35. Buettner C
    36. Haider S
    37. Bian Z
    38. Sun L
    39. Rosen CJ
    40. Zaidi M

    (2017) Blocking FSH induces thermogenic adipose tissue and reduces body fat

    Nature 546:107–112.

    https://doi.org/10.1038/nature22342

    • Google Scholar
42. 1. Luke GA
    2. Ryan MD

    (2018) Using the 2A protein coexpression system: multicistronic 2A vectors expressing gene(s) of interest and reporter proteins

    Methods in Molecular Biology 1755:31–48.

    https://doi.org/10.1007/978-1-4939-7724-6_3

    • PubMed
    • Google Scholar
43. 1. Mather JP

    (1980) Establishment and characterization of two distinct mouse testicular epithelial cell lines

    Biology of Reproduction 23:243–252.

    https://doi.org/10.1095/biolreprod23.1.243

    • PubMed
    • Google Scholar
44. 1. Matz MV
    2. Fradkov AF
    3. Labas YA
    4. Savitsky AP
    5. Zaraisky AG
    6. Markelov ML
    7. Lukyanov SA

    (1999) Fluorescent proteins from nonbioluminescent Anthozoa species

    Nature Biotechnology 17:969–973.

    https://doi.org/10.1038/13657

    • PubMed
    • Google Scholar
45. 1. Minegishi T
    2. Nakamura K
    3. Takakura Y
    4. Ibuki Y
    5. Igarashi M
    6. Minegishi T

    (1991) Cloning and sequencing of human FSH receptor cDNA

    Biochemical and Biophysical Research Communications 175:1125–1130.

    https://doi.org/10.1016/0006-291x(91)91682-3

    • PubMed
    • Google Scholar
46. 1. Miyauchi H
    2. Ohta H
    3. Nagaoka S
    4. Nakaki F
    5. Sasaki K
    6. Hayashi K
    7. Yabuta Y
    8. Nakamura T
    9. Yamamoto T
    10. Saitou M

    (2017) Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice

    The EMBO Journal 36:3100–3119.

    https://doi.org/10.15252/embj.201796875

    • PubMed
    • Google Scholar
47. 1. Nazareth L
    2. Lineburg KE
    3. Chuah MI
    4. Tello Velasquez J
    5. Chehrehasa F
    6. St John JA
    7. Ekberg JAK

    (2015) Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

    The Journal of Comparative Neurology 523:479–494.

    https://doi.org/10.1002/cne.23694

    • PubMed
    • Google Scholar
48. 1. Ohta T
    2. Miyake H
    3. Miura C
    4. Kamei H
    5. Aida K
    6. Miura T

    (2007) Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica

    Biology of Reproduction 77:970–977.

    https://doi.org/10.1095/biolreprod.107.062299

    • PubMed
    • Google Scholar
49. 1. Papadimitriou K
    2. Kountourakis P
    3. Kottorou AE
    4. Antonacopoulou AG
    5. Rolfo C
    6. Peeters M
    7. Kalofonos HP

    (2016) Follicle-Stimulating Hormone Receptor (FSHR): a promising tool in oncology?

    Molecular Diagnosis & Therapy 20:523–530.

    https://doi.org/10.1007/s40291-016-0218-z

    • Google Scholar
50. 1. Pippin JW
    2. Sparks MA
    3. Glenn ST
    4. Buitrago S
    5. Coffman TM
    6. Duffield JS
    7. Gross KW
    8. Shankland SJ

    (2013) Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease

    The American Journal of Pathology 183:542–557.

    https://doi.org/10.1016/j.ajpath.2013.04.024

    • PubMed
    • Google Scholar
51. 1. Pippin JW
    2. Glenn ST
    3. Krofft RD
    4. Rusiniak ME
    5. Alpers CE
    6. Hudkins K
    7. Duffield JS
    8. Gross KW
    9. Shankland SJ

    (2014) Cells of renin lineage take on a podocyte phenotype in aging nephropathy

    American Journal of Physiology. Renal Physiology 306:F1198–F1209.

    https://doi.org/10.1152/ajprenal.00699.2013

    • PubMed
    • Google Scholar
52. 1. Pozzobon M
    2. Piccoli M
    3. Ditadi A
    4. Bollini S
    5. Destro R
    6. André-Schmutz I
    7. Masiero L
    8. Lenzini E
    9. Zanesco L
    10. Petrelli L
    11. Cavazzana-Calvo M
    12. Gazzola MV
    13. De Coppi P

    (2009) Mesenchymal stromal cells can be derived from bone marrow CD133+ cells: implications for therapy

    Stem Cells and Development 18:497–510.

    https://doi.org/10.1089/scd.2008.0003

    • PubMed
    • Google Scholar
53. 1. Robinson LJ
    2. Tourkova I
    3. Wang Y
    4. Sharrow AC
    5. Landau MS
    6. Yaroslavskiy BB
    7. Sun L
    8. Zaidi M
    9. Blair HC

    (2010) FSH-receptor isoforms and FSH-dependent gene transcription in human monocytes and osteoclasts

    Biochemical and Biophysical Research Communications 394:12–17.

    https://doi.org/10.1016/j.bbrc.2010.02.112

    • PubMed
    • Google Scholar
54. 1. Sacchi S
    2. Sena P
    3. Degli Esposti C
    4. Lui J
    5. La Marca A

    (2018) Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium

    Journal of Assisted Reproduction and Genetics 35:1703–1712.

    https://doi.org/10.1007/s10815-018-1248-8

    • PubMed
    • Google Scholar
55. 1. Saito K
    2. He Y
    3. Yan X
    4. Yang Y
    5. Wang C
    6. Xu P
    7. Hinton AO Jr
    8. Shu G
    9. Yu L
    10. Tong Q
    11. Xu Y

    (2016) Visualizing estrogen receptor-α-expressing neurons using a new ERα-ZsGreen reporter mouse line

    Metabolism 65:522–532.

    https://doi.org/10.1016/j.metabol.2015.12.011

    • PubMed
    • Google Scholar
56. 1. Simms BA
    2. Zamponi GW

    (2014) Neuronal voltage-gated calcium channels: structure, function, and dysfunction

    Neuron 82:24–45.

    https://doi.org/10.1016/j.neuron.2014.03.016

    • PubMed
    • Google Scholar
57. 1. Simoni M
    2. Gromoll J
    3. Nieschlag E

    (1997) The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology

    Endocrine Reviews 18:739–773.

    https://doi.org/10.1210/edrv.18.6.0320

    • PubMed
    • Google Scholar
58. 1. Springer TA

    (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm

    Cell 76:301–314.

    https://doi.org/10.1016/0092-8674(94)90337-9

    • PubMed
    • Google Scholar
59. 1. Stilley JAW
    2. Christensen DE
    3. Dahlem KB
    4. Guan R
    5. Santillan DA
    6. England SK
    7. Al-Hendy A
    8. Kirby PA
    9. Segaloff DL

    (2014) FSH receptor (FSHR) expression in human extragonadal reproductive tissues and the developing placenta, and the impact of its deletion on pregnancy in mice

    Biology of Reproduction 91:74.

    https://doi.org/10.1095/biolreprod.114.118562

    • PubMed
    • Google Scholar
60. 1. Stilley JAW
    2. Segaloff DL

    (2018) Deletion of fetoplacental Fshr inhibits fetal vessel angiogenesis in the mouse placenta

    Molecular and Cellular Endocrinology 476:79–83.

    https://doi.org/10.1016/j.mce.2018.04.011

    • PubMed
    • Google Scholar
61. 1. Su J
    2. Song Y
    3. Yang Y
    4. Li Z
    5. Zhao F
    6. Mao F
    7. Wang D
    8. Cao G

    (2023) Study on the changes of LHR, FSHR and AR with the development of testis cells in Hu sheep

    Animal Reproduction Science 256:107306.

    https://doi.org/10.1016/j.anireprosci.2023.107306

    • PubMed
    • Google Scholar
62. 1. Sun L
    2. Peng Y
    3. Sharrow AC
    4. Iqbal J
    5. Zhang Z
    6. Papachristou DJ
    7. Zaidi S
    8. Zhu L-L
    9. Yaroslavskiy BB
    10. Zhou H
    11. Zallone A
    12. Sairam MR
    13. Kumar TR
    14. Bo W
    15. Braun J
    16. Cardoso-Landa L
    17. Schaffler MB
    18. Moonga BS
    19. Blair HC
    20. Zaidi M

    (2006) FSH directly regulates bone mass

    Cell 125:247–260.

    https://doi.org/10.1016/j.cell.2006.01.051

    • Google Scholar
63. 1. Tedjawirja VN
    2. Hooijer GKJ
    3. Savci-Heijink CD
    4. Kovac K
    5. Balm R
    6. de Waard V

    (2023) Inadequate detection of the FSHR complicates future research on extragonadal FSHR localization

    Frontiers in Endocrinology 14:1095031.

    https://doi.org/10.3389/fendo.2023.1095031

    • PubMed
    • Google Scholar
64. 1. Trichas G
    2. Begbie J
    3. Srinivas S

    (2008) Use of the viral 2A peptide for bicistronic expression in transgenic mice

    BMC Biology 6:40.

    https://doi.org/10.1186/1741-7007-6-40

    • PubMed
    • Google Scholar
65. 1. Wada T
    2. Becskei A

    (2017) Impact of methods on the measurement of mRNA turnover

    International Journal of Molecular Sciences 18:2723.

    https://doi.org/10.3390/ijms18122723

    • PubMed
    • Google Scholar
66. 1. Wang Y
    2. Wang F
    3. Wang R
    4. Zhao P
    5. Xia Q

    (2015) 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

    Scientific Reports 5:16273.

    https://doi.org/10.1038/srep16273

    • PubMed
    • Google Scholar
67. 1. Wang S
    2. Zhu Q
    3. Liang G
    4. Franks T
    5. Boucher M
    6. Bence KK
    7. Lu M
    8. Castorena CM
    9. Zhao S
    10. Elmquist JK
    11. Scherer PE
    12. Horton JD

    (2021) Cannabinoid receptor 1 signaling in hepatocytes and stellate cells does not contribute to NAFLD

    Journal of Clinical Investigation 131:JCI152242.

    https://doi.org/10.1172/JCI152242

    • Google Scholar
68. 1. Xiao L
    2. McCann JV
    3. Dudley AC

    (2015) Isolation and culture expansion of tumor-specific endothelial cells

    Journal of Visualized Experiments 01:e53072.

    https://doi.org/10.3791/53072

    • PubMed
    • Google Scholar
69. 1. Xiong J
    2. Kang SS
    3. Wang Z
    4. Liu X
    5. Kuo T-C
    6. Korkmaz F
    7. Padilla A
    8. Miyashita S
    9. Chan P
    10. Zhang Z
    11. Katsel P
    12. Burgess J
    13. Gumerova A
    14. Ievleva K
    15. Sant D
    16. Yu S-P
    17. Muradova V
    18. Frolinger T
    19. Lizneva D
    20. Iqbal J
    21. Goosens KA
    22. Gera S
    23. Rosen CJ
    24. Haroutunian V
    25. Ryu V
    26. Yuen T
    27. Zaidi M
    28. Ye K

    (2022) FSH blockade improves cognition in mice with Alzheimer’s disease

    Nature 603:470–476.

    https://doi.org/10.1038/s41586-022-04463-0

    • PubMed
    • Google Scholar
70. 1. Young AP
    2. Jackson DJ
    3. Wyeth RC

    (2020) A technical review and guide to RNA fluorescence in situ hybridization

    PeerJ 8:e8806.

    https://doi.org/10.7717/peerj.8806

    • PubMed
    • Google Scholar
71. 1. Zhu L-L
    2. Blair H
    3. Cao J
    4. Yuen T
    5. Latif R
    6. Guo L
    7. Tourkova IL
    8. Li J
    9. Davies TF
    10. Sun L
    11. Bian Z
    12. Rosen C
    13. Zallone A
    14. New MI
    15. Zaidi M

    (2012a) Blocking antibody to the β-subunit of FSH prevents bone loss by inhibiting bone resorption and stimulating bone synthesis

    PNAS 109:14574–14579.

    https://doi.org/10.1073/pnas.1212806109

    • PubMed
    • Google Scholar
72. 1. Zhu J
    2. Jankovic D
    3. Oler AJ
    4. Wei G
    5. Sharma S
    6. Hu G
    7. Guo L
    8. Yagi R
    9. Yamane H
    10. Punkosdy G
    11. Feigenbaum L
    12. Zhao K
    13. Paul WE

    (2012b) The transcription factor T-bet Is induced by multiple pathways and prevents an endogenous Th2 Cell Program during Th1 Cell Responses

    Immunity 37:660–673.

    https://doi.org/10.1016/j.immuni.2012.09.007

    • Google Scholar
73. 1. Zhu L-L
    2. Tourkova I
    3. Yuen T
    4. Robinson LJ
    5. Bian Z
    6. Zaidi M
    7. Blair HC

    (2012c) Blocking FSH action attenuates osteoclastogenesis

    Biochemical and Biophysical Research Communications 422:54–58.

    https://doi.org/10.1016/j.bbrc.2012.04.104

    • PubMed
    • Google Scholar
74. 1. Zhu X
    2. Ricci-Tam C
    3. Hager ER
    4. Sgro AE

    (2023) Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

    PLOS ONE 18:e0281211.

    https://doi.org/10.1371/journal.pone.0281211

    • Google Scholar

Author details

1. Hongqian Chen

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization

   Contributed equally with

   Hui-Qing Fang

   Competing interests

   No competing interests declared

2. Hui-Qing Fang

   1. Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China
   2. Department of Dentistry, The 980th Hospital of the PLA Joint Logistic Support Force, Shijiazhuang, China

   Contribution

   Data curation, Formal analysis, Investigation

   Contributed equally with

   Hongqian Chen

   Competing interests

   No competing interests declared

3. Jin-Tao Liu

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

4. Shi-Yu Chang

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

5. Li-Ben Cheng

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Ming-Xin Sun

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Jian-Rui Feng

   Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

8. Ze-Min Liu

   1. Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China
   2. Shanxi Medical Universityersity, The Second Hospital, University Shanxi Medical University, Taiyuan, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4810-9200

9. Yong-Hong Zhang

   1. Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China
   2. Shanxi Medical Universityersity, The Second Hospital, University Shanxi Medical University, Taiyuan, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

10. Clifford J Rosen

    Maine Medical Center Research Institute, Scarborough, United States

    Contribution

    Writing - original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3436-8199

11. Peng Liu

    Laboratory of Bone and Adipose Biology, Shanxi Medical University, Taiyuan, China

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    liupossible@gmail.com

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0001-5607-2482

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